JP5690359B2 - Imaging apparatus and imaging method - Google Patents

Imaging apparatus and imaging method Download PDF

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JP5690359B2
JP5690359B2 JP2013011736A JP2013011736A JP5690359B2 JP 5690359 B2 JP5690359 B2 JP 5690359B2 JP 2013011736 A JP2013011736 A JP 2013011736A JP 2013011736 A JP2013011736 A JP 2013011736A JP 5690359 B2 JP5690359 B2 JP 5690359B2
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light
sample
imaging
image
distribution
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JP2013228361A5 (en
JP2013228361A (en
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藤本 博己
博己 藤本
小林 正嘉
正嘉 小林
三宅 孝志
孝志 三宅
森脇 三造
三造 森脇
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株式会社Screenホールディングス
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    • HELECTRICITY
    • H04ELECTRIC COMMUNICATION TECHNIQUE
    • H04NPICTORIAL COMMUNICATION, e.g. TELEVISION
    • H04N7/00Television systems
    • H04N7/18Closed circuit television systems, i.e. systems in which the signal is not broadcast
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251Colorimeters; Construction thereof
    • G01N21/253Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/255Details, e.g. use of specially adapted sources, lighting or optical systems
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/063Illuminating optical parts
    • G01N2201/0631Homogeneising elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/063Illuminating optical parts
    • G01N2201/0634Diffuse illumination
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/067Electro-optic, magneto-optic, acousto-optic elements

Description

  The present invention relates to an image pickup apparatus and an image pickup method for picking up an image of a sample formed by injecting a fluid into a recess, and more particularly to a technique for adjusting irradiation light for the illumination.

  In medical and biological science experiments, for example, a liquid or gel-like fluid (for example, a culture solution or a medium) is injected into each well of a plate-like instrument provided with a large number of depressions, also called wells. Here, observing and measuring a cultured cell or the like as a sample is performed. Such an instrument is called, for example, a microplate or a microtiter plate. In recent years, a sample is imaged with a CCD camera or the like and converted into data, and the image data is subjected to various image processing and used for observation and analysis.

  In this case, even if the distribution of the amount of illumination light applied to the sample is uniform, the amount of light incident on the sample contents (cells, etc.) due to the refraction of light by the meniscus on the surface (liquid surface) of the sample is small. Depending on the position, the image may become non-uniform, and uneven brightness due to this may occur in the image. As a conventional technique that focuses on such a problem, for example, in the technique described in Patent Document 1, illumination is performed by reconstructing an image of one well from partial images that are captured a plurality of times with different light incident directions. Elimination of image density unevenness due to light unevenness is attempted.

U.S. Pat. No. 7,718,131

  The above prior art merely switches the incident direction of light, and does not solve the shortage of light quantity at a portion where incident light is difficult to reach, for example, a peripheral portion of a recess. Accordingly, the amount of light incident on the sample contents has not been made uniform. Such unevenness in the amount of incident light causes unevenness in brightness in the image of the sample contents (cells, etc.). For example, when performing analysis such as cell detection and type discrimination automatically by image analysis, the brightness of the analysis object such as cells contained in the image is used as important information. The accuracy of such analysis is lowered, and the above-described conventional technique cannot cope with this problem.

  The present invention has been made in view of the above problems, and provides a technique capable of reducing density unevenness in an image caused by irradiation light in a technique for capturing an image of a sample formed by injecting a fluid into a recess. The purpose is to do.

In order to achieve the above object, an aspect of the imaging apparatus according to the present invention is configured to hold a sample holding plate formed with a recess capable of holding a liquid in a substantially horizontal state, and to be held by the holding unit. A light irradiating means for causing light to enter the surface of the sample formed by injecting a fluid into the recess from above the sample holding plate; and an imaging means for imaging the sample and obtaining an image of the sample; Control means for controlling the light quantity distribution of light incident on the surface of the sample from the light irradiating means, wherein the control means sets the light quantity distribution in a predetermined standard state in advance and the imaging means controls the sample. based on the luminance distribution in the background image obtained by removing a predetermined spatial frequency than high frequency components from the pre-captured image captured, it sets the light amount distribution of light to be incident on the surface of the sample, the image pickup means , Under light irradiation at the set light amount distribution by the control means is characterized by acquiring an image of the sample.

According to another aspect of the present invention, there is provided an imaging method for imaging a sample formed by injecting a fluid into a recess provided in a sample holding plate, wherein the sample is held substantially horizontally in order to achieve the above object. A pre-imaging step of irradiating light with a light amount distribution in a predetermined standard state from above the sample holding plate toward the surface of the sample, imaging the sample and obtaining a pre-captured image, and the pre-captured image A luminance distribution detection step for detecting a luminance distribution of a background image from which a high frequency component equal to or higher than a predetermined spatial frequency is removed, and a light amount distribution for setting a light amount distribution of light incident on the sample based on a detection result in the detection step The method includes a setting step and an image acquisition step of acquiring an image of the sample under light irradiation with the light amount distribution set in the light amount distribution setting step.

In the invention configured as described above, the background image obtained by removing the high-frequency component from the pre-captured image obtained by actually capturing the sample to be imaged after the light amount distribution of the irradiation light is once set to a predetermined standard state. Based on the luminance distribution at, light irradiation conditions for acquiring an image of the sample are set. In this way, by grasping the luminance distribution from an image captured in advance with a predetermined light amount distribution, the sample can be imaged while irradiating the inside of the sample with light having a uniform light amount distribution. Therefore, according to the present invention, it is possible to reduce the luminance unevenness of the image due to the light amount unevenness of the irradiation light, and obtain a high-quality image that can be suitably used for image analysis or the like. The sample to be imaged includes cells and the like distributed in the fluid, but such an image of cells and the like can be an error factor when detecting unevenness in the amount of irradiation light. Therefore, it is possible to eliminate the influence by removing a high spatial frequency component, and to detect the luminance distribution in the remaining background image, thereby setting the light amount with higher accuracy.

  In this type of sample, the shape of the unevenness on the surface may not be constant for each sample due to, for example, variations in the way of injecting the fluid and wettability to the well wall surface. Since the light quantity distribution is set based on the pre-imaging result of the sample that is the actual imaging target, it is possible to cope with such sample variations.

In these inventions, for example, as the amount of incident light as the position low brightness in bar click background image is increased, it is possible to set the light intensity distribution of the illumination light. Ba background concentrations by so as to be incident more light for low position luminance in the image, it is possible to obtain the image with less luminance unevenness.

Another aspect of the imaging device includes a holding means for holding a sample holding plate recess capable of holding is formed a liquid material in a substantially horizontal state, above the sample holding plate held by said holding means A light irradiating means for emitting light toward the surface of the sample in which a fluid is injected into the recess, and a light quantity distribution of the light emitted from the light irradiating means and incident on the surface of the sample. A control means for equalizing a light quantity distribution of light that passes through the surface of the sample and is incident on the inside of the sample, and images the sample under light irradiation with the light quantity distribution controlled by the control means. Ru and an imaging means for obtaining an image of the sample Te.

In such a configuration , the light quantity distribution of incident light on the surface of the sample is not made uniform, but the light quantity distribution of incident light is controlled so as to be uniform. In other words, in anticipation that the light amount distribution changes due to refraction at the sample surface, a light amount distribution that compensates for this is given to the incident light on the sample surface in advance. This also makes it possible to obtain an image with less luminance unevenness by making the amount of incident light uniform with respect to the contents of the sample.

  The light irradiation means in the image pickup apparatus according to the present invention includes, for example, a light source and a transmitted light amount adjustment unit that is disposed between the light source and the sample and can set the amount of light transmission for each position, and is a control means. Can control the transmitted light amount adjustment unit. According to such a configuration, since the light amount distribution of the light finally incident on the sample is adjusted by the transmitted light amount adjusting unit provided between the light source and the sample, the light amount distribution is finely adjusted in the light source itself. No function is required, and various light emitting devices can be used as the light source. Also, the inherent light quantity distribution of the light source itself does not affect the image quality.

  In this case, the transmitted light amount adjustment unit may be able to set different transmission amounts for a plurality of wavelength components included in the light from the light source. In this way, it is possible to acquire a wider variety of images by controlling the spectral distribution of light incident on the sample. For example, the wavelength of irradiation light can be switched according to the purpose of imaging.

  For example, a liquid crystal shutter can be used as the transmitted light amount adjustment unit that realizes these functions. Low-priced products that can adjust the amount of transmitted light in small pixel units are already widely used as liquid crystal shutters, and it is possible to set the light amount distribution of the present invention by applying such products. is there.

  Further, for example, the light irradiation unit may include a light source in which a plurality of light emitting modules are arranged, and the control unit may individually control the light emission amounts of the plurality of light emitting modules. According to such a configuration, the light quantity distribution itself of the light emitted from the light source can be changed. For such an application, for example, an array of many light emitting elements such as an LED (Light Emitting Diode) array can be used as a light source.

  Further, for example, the light irradiation means includes a light source, and a plurality of reflection mirrors that reflect light from the light source to be incident on the surface of the sample and can change the reflection direction independently of each other, and the control means includes a plurality of control mirrors. The reflection direction of each of the reflection mirrors may be controlled. It is also possible to adjust the light quantity distribution of light incident on the sample by changing the reflection direction of light from the light source. For such an application, for example, a device such as DMD (Digital Mirror Device) in which a large number of minute reflecting mirrors are arranged can be used.

  In these inventions, for example, a sample image may be acquired using a line sensor that scans and moves relative to the sample holding plate. As a line sensor for imaging purposes, a high-resolution sensor that can be suitably applied to the application of the present invention has been commercialized, and by scanning this with respect to the sample held on the sample holding plate, A high-quality two-dimensional image with high resolution can be acquired.

  In this case, the light irradiation means may be fixed to the sample holding plate, or may be moved relative to the sample holding plate integrally with the line sensor as the line sensor scans. . In any configuration, it is possible to perform imaging with a sufficient amount of light by irradiating the imaging target region by the line sensor with light. As a configuration in which the light irradiating means is not moved, for example, a surface light source (including a light diffusing means) that can irradiate the entire upper surface of the recess can be used.

  In addition, when moving together with the line sensor, the set light amount distribution can be realized by changing the light amount distribution of the light incident on the surface of the sample in synchronization with the relative movement of the light irradiation means with respect to the sample holding plate. . As a configuration for realizing such a function, for example, a line light source (for example, an LED array) in which a large number of light emitting elements are arranged along the arrangement direction of the image pickup elements in the line sensor is used. It is sufficient to control them.

  Furthermore, in the imaging method according to the present invention, when imaging is performed for each of the samples held in each of the plurality of recesses provided in the sample holding plate, a preliminary imaging step, a luminance distribution detection step, and a light amount distribution setting step It is desirable to perform the image acquisition process for each sample. Since the surface condition of the sample and the positional relationship with the light irradiation means differ from sample to sample, high-quality images that do not depend on sample variation can be stabilized by setting the irradiation light intensity distribution for each sample and capturing images. Can be taken.

  According to the present invention, the light whose distribution of light intensity is adjusted in advance is applied in consideration of refraction at the sample surface so that uniform light is incident on the contents of the sample in which the fluid is injected into the recess. In this state, an image of the sample is acquired. For this reason, it is possible to reduce the density unevenness of the image due to the light quantity unevenness of the irradiation light, and to obtain a high quality image that can be suitably used for image analysis or the like.

1 is a diagram illustrating a schematic configuration of a first embodiment of an imaging apparatus according to the present invention. It is a figure which shows the structure of a microplate in detail. It is a figure which shows the two imaging aspects which the apparatus of 1st Embodiment can take. It is a flowchart which shows the imaging operation in 1st Embodiment. It is a figure which shows the calculation model of irradiation light quantity distribution. It is a figure which shows the principal part of 2nd Embodiment of the imaging device concerning this invention. It is a flowchart which shows the imaging operation in 2nd Embodiment. It is a figure explaining the view of a light emission profile. It is a figure which shows the principal part of 3rd Embodiment of the imaging device concerning this invention. It is a figure which shows the principal part of 4th Embodiment of the imaging device concerning this invention. It is a flowchart which shows an example of the preparation method of the transmitted light amount adjustment member in 4th Embodiment. It is a figure which shows the example of a setting of the line segment in 4th Embodiment. It is a figure for demonstrating the calculation principle of the luminance value of the pixel in a well. It is a figure which shows the principal part of 5th and 6th embodiment of the imaging device concerning this invention. It is a figure which shows the example which adjusts light quantity distribution between a well and an imaging unit.

  FIG. 1 is a diagram showing a schematic configuration of a first embodiment of an imaging apparatus according to the present invention. The imaging apparatus 1 is brought into contact with the peripheral edge of the lower surface of a sample (microplate) M in which a plurality of, for example, 96 (12 × 8 matrix array) wells W are formed, so that the microplate M is in a substantially horizontal state. The holder 11 to hold | maintain, the illumination part 12 provided in the upper part of this holder 11, the imaging unit 13 provided in the lower part of the holder 11, and the control part 10 which manages these and performs a predetermined | prescribed operation | movement are provided. ing. For the following description, coordinate axes are set as shown in FIG. The XY plane is a horizontal plane, and the Z axis is a vertical axis.

  The diameter and depth of each well W in the microplate M is typically about several millimeters, and each well W is injected with a liquid such as a culture solution, a medium, a reagent, or the like (only a part is shown). Yes. The number of wells and the size of the microplate targeted by the imaging apparatus 1 are not limited to these and are arbitrary.

  The illumination unit 12 is controlled by a light source control unit 112a provided in the control unit 10, and emits light from above toward the microplate M held by the holder 11 in accordance with a control command from the light source control unit 112a. 12a. The light source 12 a is a surface light source that has a planar size that is equal to or larger than the planar size of the microplate M held by the holder 11 and emits light with a substantially uniform light amount distribution from the lower surface thereof. The light emitted from the light source 12a is visible light, and white light is particularly preferable. As such a light source, a fluorescent light source, an EL (Electroluminescence) light source, a multi-LED array, and the like can be used.

  On the lower surface of the light source 12a, a transmitted light amount adjustment unit 12b made of, for example, a liquid crystal shutter is disposed, and the light amount distribution of light emitted from the light source 12a toward the microplate M is controlled. More specifically, the transmitted light amount adjustment unit 12b opens and closes the optical path of light incident from one main surface side and transmitted to the other main surface side in units of minute cells, so that the one main surface side to the other main surface. Adjust the amount of light transmitted to the side. The opening and closing of the shutter is controlled for each cell by a shutter control unit 112b provided in the control unit 10. Thereby, for example, the larger the area in which the shutter is opened in the unit area, the greater the amount of light transmitted in that region. In this way, by controlling the light transmission amount for each position, the light amount distribution of the light incident on the microplate M can be finely controlled.

  In addition, as the illumination part 12 which consists of a surface light source and a liquid-crystal shutter as mentioned above, it is possible to use the liquid crystal display panel already marketed, for example. That is, the backlight provided in the liquid crystal display panel can function as the light source 12a, and the liquid crystal unit including the drive circuit can function as the transmitted light amount adjusting unit 12b. And the transmitted light quantity distribution of various patterns is realizable similarly to displaying a figure pattern on a display panel. Furthermore, if a display panel capable of color display is used, it is possible to set transmission patterns for RGB wavelength components independently. In the case where the resolution of the image sensor is high and the color distribution between the color pixels is conspicuous, an appropriate diffusion plate may be combined with the display panel to increase the degree of light scattering. The surface light source may be not only a shutter type such as a liquid crystal but also a surface light emitting type such as an organic EL panel.

  The illumination unit 12 configured as described above collectively irradiates the plurality of wells W formed on the microplate M with the illumination light L as the illumination light. In order to facilitate the placement of the microplate M on the holder 11 and the removal of the microplate M from the holder 11, it is desirable that the illumination unit 12 be retracted from the upper portion of the holder 11. . For this purpose, it is desirable to support the illumination unit 12 by a movable member such as a movable arm or a hinge (not shown).

  The imaging unit 13 functions as a camera that captures an image of the microplate M by receiving transmitted light Lt emitted from the illumination unit 12 and transmitted below the microplate M held by the holder 11. is there. The imaging resolution is, for example, about 2400 dpi (dots per inch). The imaging unit 13 is connected to a camera driving mechanism 113 provided in the control unit 10, and the camera driving mechanism 113 scans and moves the imaging unit 13 in the horizontal plane along the lower surface of the microplate M held by the holder 11. Let That is, in this embodiment, the imaging unit 13 can be scanned and moved along the lower surface of the microplate M.

  Image data picked up by the image pickup unit 13 is given to the image processing unit 114. The image processing unit 114 appropriately performs image processing on the image data from the imaging unit 13 or executes predetermined calculation processing based on the image data. Data before and after processing is stored and saved in the storage unit 115 as necessary. Further, the detection processing unit 116 performs a predetermined detection process based on the image data given from the image processing unit 114, and detects a characteristic part included in the image. This detection process is a process for detecting areas in which the optical characteristics are different from the surrounding area in the image by analyzing luminance data of the image, for example, and by calculating a feature amount for the area, It is possible to classify the origin and type of the area. Since various techniques are known for the process of identifying and detecting a part having a certain feature from the image and the feature amount suitable for such a process, detailed description thereof is omitted here.

  The detection result by the detection processing unit 116 is also stored in the storage unit 115. Further, the image processing unit 114 performs image processing based on the detection result by the detection processing unit 116 as necessary. Then, the image data subjected to appropriate image processing is given to a display unit 118 having display means such as a liquid crystal display, and the display unit 118 displays an image corresponding to the given image data and presents it to the user. . Further, the imaging apparatus 1 includes an input receiving unit 117 for receiving an operation instruction input from the user regarding the contents of image processing, the display mode, and the like. The input receiving unit 117 is an input receiving unit such as a keyboard, a mouse, and a touch pad, or a combination of them. The input receiving unit 117 receives an instruction input from the user, and the control unit 10 reflects this in the operation of the apparatus. The function desired by the user is realized.

  The imaging apparatus 1 is configured such that a fluid (in this specification, a liquid, a gel-like or semi-fluid solid, and a fluid having a fluidity such as soft agar is held in each well W. And an optical image of an object to be imaged such as cells contained therein, or having a predetermined optical characteristic from the optical image, more specifically, in the well W The present invention can be applied to an application in which a specific portion having an optical characteristic different from that of a retained liquid or the like is detected using a difference in the optical characteristic. For example, it can be suitably used for the purpose of imaging a cell or a cell clump (spheroid) in a culture solution or a medium as an imaging object, or automatically detecting such a cell or the like by image processing.

  FIG. 2 is a diagram showing the structure of the microplate in more detail. As shown in FIG. 2 (a), the microplate M has through-holes M1 having a substantially cylindrical side surface (more precisely, tapered with a cross-sectional area gradually decreasing toward the bottom surface) with regular pitches. The upper plate M2 is arranged in a two-dimensional matrix, and the lower sheet M3 is attached to the lower surface of the upper plate M2 so as to close each through hole M1.

  The lower surface sheet M3 is closely attached to the lower surface of the upper plate M2, and the liquid can be held in a space surrounded by the side surface of the through hole M1 of the upper plate M2 and the lower surface sheet M3. That is, this space functions as a well W for holding a fluid, and the side surface of the through hole M1 forms the side wall surface of the well W, and the lower surface sheet M3 forms the bottom surface of the well W. The lower surface sheet M3 is a sheet formed of a transparent resin, for example, a PET (polyethylene terephthalate) resin.

  Consider a case where light is incident from above in a state where a fluid (for example, a liquid) is injected into the well W of the microplate M configured as described above. At this time, as shown in FIG. 2B, the surface (liquid level) S of the injected fluid forms a meniscus. When light L having a uniform in-plane distribution is incident on the well W from above in this state, the incident light L travels substantially straight and enters the fluid near the center of the well W where the liquid level S is substantially horizontal. On the other hand, the traveling direction of the light incident on the liquid surface near the side wall surface of the well W is bent due to refraction at the liquid surface. Due to this, the illumination light is difficult to reach at the peripheral edge P near the side wall surface of the well bottom surface, and as shown in the lower part of FIG. .

  Such a distribution of the amount of incident light means that, for example, the amount of illumination light incident on an object such as a cell cultured on the bottom of the well varies from position to position. For this reason, even if the images of the same object, the luminance varies depending on the position in the well W. This can cause an error in the process of analyzing the object with the brightness of the imaged object as significant information.

  Therefore, in this embodiment, as described above, the illumination unit 12 capable of controlling the irradiation light amount distribution is provided. In the imaging operation described later, the illumination light quantity distribution at the time of imaging is set so that uniform light is incident on the well bottom surface based on the luminance distribution in the pre-captured image obtained by imaging the sample to be imaged in advance. The imaging is performed under the illumination conditions.

  The surface state of the fluid injected into the well W varies from sample to sample due to the viscosity of the fluid, the wettability with respect to the well wall surface, the work variation during injection, and the like. Due to this, the distribution of the incident light quantity on the bottom surface of the well can be different for each sample. The technical idea of the present embodiment can cope with the different liquid level states for each sample as described above, but here, in order to facilitate understanding, as shown in FIG. It is assumed that a meniscus whose surface is convex downward is formed.

  FIG. 3 is a diagram showing two imaging modes that the apparatus of the present embodiment can take. Here, an example in which imaging is performed for one well W will be described, but the concept is the same even when imaging is performed for a plurality of wells at once. In the first mode shown in FIG. 3A, the substantially uniform illumination light L from the illumination unit 12 enters from above the well W that is the imaging target. Below the well W, an imaging optical system 131 and a two-dimensional imaging element 132 in which a large number of fine light receiving elements are two-dimensionally arranged are sequentially arranged. These constitute the imaging unit 13, but in FIG. 3A, a reference numeral 13 a is attached to distinguish the imaging unit having the configuration from others. As the two-dimensional image sensor 132, a CCD sensor, a CMOS sensor, or the like can be used. Here, the imaging optical system 131 is shown as a single lens, but it may be composed of a plurality of lenses.

  The transmitted light from the bottom surface of the well W is converged by the imaging optical system 131 on the light receiving surface of the two-dimensional image sensor 132 disposed below the well. As a result, an image of an object (cell or the like) distributed on the bottom surface of the well and in the vicinity thereof is formed on the light receiving surface of the two-dimensional image sensor 132, and an image of the well is acquired by the two-dimensional image sensor 132. This aspect can be said to be a combination of a two-dimensional pattern illumination unit and a two-dimensional pattern image sensor.

  On the other hand, in the image pickup unit 13b of the second mode shown in FIG. 3B, a one-dimensional image pickup element (line sensor) 133 in which a number of fine light receiving elements are arranged one-dimensionally is arranged below the imaging optical system 131. Is done. Then, the two-dimensional image of the well W is acquired by performing the imaging while the one-dimensional imaging element 133 scans and moves in the direction Ds orthogonal to the longitudinal direction (light receiving element arrangement direction) as in the first aspect. Is done. This aspect is a combination of a two-dimensional pattern illumination unit and a one-dimensional pattern image sensor. Even if the imaging unit 13 has the configuration of any one of the above-described configurations, the imaging operation described below can be applied, and the operational effects obtained thereby are basically the same.

  FIG. 4 is a flowchart showing the imaging operation in the first embodiment. After the microplate M holding the sample in the well W is set in the holder 11 by the operator, the control unit 10 controls each part of the apparatus and executes the operation of FIG. 4 to acquire an image of the well W containing the sample. Is done. First, the liquid crystal shutter of the transmitted light amount adjustment unit 12b is set to a fully open state, that is, a state in which light from the light source 12a is allowed to pass at all positions in the plane (step S101). Thereby, substantially uniform light emitted from the light source 12a is incident on the surface of the sample held in the well W as it is. The light quantity distribution at this time is set to a standard state which becomes a reference for later operation.

  In this state, an imaging operation (pre-scan operation) by the imaging unit 13 is executed (step S102). Hereinafter, the image obtained at this time is referred to as a “pre-captured image”. In the pre-captured image, luminance unevenness due to the refraction of the illumination light on the sample surface occurs, and this is not necessarily suitable for various analyzes as it is. In this embodiment, a pre-captured image is used as a sample for optimizing the illumination condition in the subsequent main scan operation.

  Subsequently, the pre-captured image is smoothed (step S103). That is, a relatively high spatial frequency component included in the image is removed by appropriate filter processing or the like. The pre-captured image includes an image of a cell or the like in the sample, but what is needed at this point is information on the luminance distribution caused by the illumination light, and information on the cell and the like is unnecessary. By performing smoothing corresponding to the assumed size of cells and the like, a background image obtained by removing the image of cells and the like from the image can be obtained.

  The luminance in the background image thus obtained is obtained for each pixel, and the luminance distribution in the image is detected (step S104). The luminance distribution detected here indicates the distribution of the amount of light incident on the bottom of the well. Next, in order to obtain a uniform incident light amount distribution in actual imaging, a distribution of the irradiation light amount that cancels the luminance distribution thus detected is calculated (step S105).

  FIG. 5 is a diagram showing a calculation model of the irradiation light amount distribution. When the sample surface has a downwardly convex meniscus as shown in FIG. 2B, it is assumed that a luminance distribution is obtained in which the luminance is high at the central portion of the well and low at the peripheral portion. This reflects the amount of light incident on the bottom of the well. At this time, the light itself emitted from the light source 12a is substantially uniform in the plane. Therefore, as shown by the alternate long and short dash line in FIG. 5A, if the amount of light incident on the sample surface corresponding to the portion with high luminance is suppressed in advance, the light amount distribution on the well bottom surface is brought closer to a more uniform state. Is possible. Such adjustment of the light amount distribution can be realized by controlling the transmission pattern in the transmitted light amount adjustment unit 12b.

  By adjusting the irradiation light amount distribution as described above, even if there is a slight light amount distribution (shading) in the emitted light itself from the light source 12a, this can be canceled. Similarly, luminance unevenness due to the unevenness of the sample surface can be canceled together.

  According to the above principle, the light quantity distribution approaches uniform, but the overall light quantity decreases and the image becomes dark. If uniform brightness can be ensured, the darkness of the entire image itself is not considered to be a problem. Depending on the cell type, the state of the cell itself may change due to strong light stimulation. Therefore, it can be considered that the adjustment method of reducing the amount of incident light in such a bright portion is practically sufficient. On the other hand, for applications that require more brightness, measures to increase the amount of light emitted from the light source 12a are executed as necessary, as shown by a two-dot chain line in FIG. May be.

  FIG. 5B schematically shows the light amount distribution at each position on the optical path of the illumination light. The amount of light L1 emitted from the light source 12a has a uniform (solid line) or some distribution (broken line) as shown in the upper right balloon box. On the other hand, the light transmittance in the transmitted light amount adjustment unit 12b set from the pre-captured image is a distribution obtained by inverting the luminance distribution in the pre-captured image as shown in the upper left balloon box.

  As a result, the light amount distribution of the light L2 that has passed through the transmitted light amount adjusting unit 12b increases as the region of the pre-captured image has a lower luminance as shown in the middle right balloon box. When such light L2 is incident on the sample in the well W, the light amount of the light L3 incident on the bottom surface of the well becomes substantially uniform regardless of the position, as shown in the lower right blowing box.

  In this way, by controlling the transmitted light amount adjustment unit 12b based on the luminance distribution in the pre-captured image and adjusting the light amount distribution of the illumination light incident on the well W, the light amount distribution of the light incident on the well bottom surface is appropriately adjusted. It is possible to set. That is, the light amount distribution on the sample surface for making the light amount distribution uniform on the bottom of the well may be calculated, and the transmission pattern of the transmitted light amount adjustment unit 12b for realizing it may be obtained.

  Returning to FIG. 4, the description of the imaging operation will be continued. By controlling each cell of the liquid crystal shutter of the transmitted light amount adjusting unit 12b based on the above principle, the light amount distribution of the light incident on the bottom surface of the well is made uniform (step S106). In this state, an image of the well W including an object such as a cell can be acquired by performing imaging (main scan operation) by the imaging unit 13 (step S107).

  In the imaging operation configured as described above, imaging is performed in a state in which substantially uniform light is irradiated on the object distributed on the bottom surface of the well, so that there is no luminance unevenness caused by refraction of illumination light on the sample surface. Images can be acquired. By using the image thus obtained as an output image for use in various types of analysis, in this embodiment, it is possible to provide a user with a good quality image with little luminance unevenness caused by illumination light.

  When imaging a plurality of wells W, it is desirable to set the irradiation light amount distribution for each well. In this case, one well may be selected and a series of processes shown in FIG. 4 may be performed, and after completion of the process, a target well may be newly selected and the above process may be repeated. Alternatively, a plurality of wells may be stored in the imaging field of view, and a pre-scan operation may be performed at once, a luminance distribution of a background image may be obtained for each well, and an irradiation light amount distribution may be set before performing the main scan operation. Good.

  Next, a second embodiment of the imaging apparatus according to the present invention will be described. In the imaging apparatus 1 according to the first embodiment described above, the illumination unit 12 disposed above the microplate M includes the surface light source 12a having a substantially uniform light amount distribution. On the other hand, the imaging apparatus according to the second embodiment to be described below has a linear light source in which light emitting elements are arranged in a line, and the whole well W is illuminated by scanning and moving the light source with respect to the well W. Light is incident. Since the apparatus configuration excluding this point and the basic operation of each part are the same as those of the first embodiment, the same components are denoted by the same reference numerals and description thereof will be omitted.

  FIG. 6 is a diagram showing a main part of a second embodiment of the imaging apparatus according to the present invention. As shown in FIG. 6A, in this embodiment, a rod-like illumination unit 22 that emits light L21 toward the upper surface of the microplate M is provided above the microplate M held by the holder 11. Yes. The imaging unit 13 includes the one-dimensional imaging element 133 illustrated in FIG. 3B, and the longitudinal direction of the illumination unit 22 is parallel to the arrangement direction of the light receiving elements in the one-dimensional imaging element 133. Yes. That is, this aspect is a combination of a one-dimensional pattern illumination unit and a one-dimensional pattern image sensor.

  The illumination unit 22 scans and moves in the direction Ds with respect to the microplate M integrally with the one-dimensional image sensor 133 in synchronization with the scanning movement of the one-dimensional image sensor 133 with respect to the microplate M. That is, in this embodiment, a two-dimensional image is acquired by changing the relative position of the one-dimensional image sensor 133 with respect to the well W while capturing an image of a portion of the well W that faces the one-dimensional image sensor 133. The illumination light from the illumination unit 22 is intensively applied to the part imaged by the one-dimensional image sensor 133 at each time.

  As shown in FIG. 6B, the illumination unit 22 is called, for example, an LED array, a bar LED, or the like in which a large number of minute light emitting elements (for example, LEDs) 221 are arranged in the longitudinal direction of the rod-shaped base member 222. Things can be used. Each light emitting element 221 is individually controlled to be turned on by the light source control unit 122. In other words, the light source control unit 122 can individually control the lighting timing and the light emission amount of each light emitting element 221.

  In addition, about the many light emitting elements 221 provided in the illumination part 22, it is not necessary for each one to carry out lighting control completely independently. That is, lighting control may be performed on a group basis by setting several adjacent light emitting elements as one group. Further, as lighting control modes, lighting ON / OFF or the amount of light emission can be adjusted. As control targets, lighting and extinguishing timings, applied voltage, lighting duty in intermittent lighting, and the like can be used.

  In the illumination unit 22 configured in this way, the light amount distribution in the arrangement direction of the light emitting elements 221 can be arbitrarily set by individually controlling the lighting of the light emitting elements 221 arranged in a row. In addition, by changing the lighting mode of each light emitting element 221 in synchronization with the scanning movement of the illumination unit 22, the light amount distribution in the scanning movement direction Ds orthogonal to the arrangement direction of the light emitting elements 221 can be arbitrarily changed. it can. Thus, by combining the temporal change in the light emission amount of one light emitting element 221 and making the light emission amount different among the plurality of light emitting elements 221, the light amount incident on the well W (more Strictly speaking, it is possible to arbitrarily set a two-dimensional distribution).

  In addition, as shown in FIG. 6C, the illumination unit 22 is arranged above a large number of light emitting elements 223 that are individually controlled to be lighted and the microplate M, and an array of light receiving elements in the one-dimensional imaging element 133. A plurality of light projecting members 224 arranged in a row in the same direction as the direction and emitting light toward the upper surface of the microplate M, and a light guide made of, for example, an optical fiber that optically connects the light projecting members 224 therebetween. A member 225 may be provided. Even with such a configuration, the same effects as described above can be obtained.

  FIG. 7 is a flowchart showing an imaging operation in the second embodiment. In the imaging operation of this embodiment, first, the illumination unit 22 and the one-dimensional imaging element 133 are positioned at a predetermined initial position (one end of the imaging area) (step S201). Then, as a standard state, the light emission amount of each light emitting element 221 is set to the same value (step S202). Specifically, the drive voltage applied to each light emitting element 221 is set to the same value. Even if the same voltage is applied, it is conceivable that the light emission amount varies for each light emitting element 221, but this can be canceled as in the light amount distribution of the light source 12a in the first embodiment. Shall be ignored.

  Subsequently, as in the first embodiment, acquisition of a pre-captured image by a pre-scan operation (step S203), smoothing of the pre-captured image (step S204), and detection of the luminance distribution of the smoothed background image (step S205) ) And the calculation of the irradiation light amount distribution for canceling this (step S206). Based on the irradiation light amount distribution thus obtained, a light emission profile that defines the lighting mode of the light emitting element 221 is set for each light emitting element 221 (or for each group of light emitting elements) (step S207).

  FIG. 8 is a diagram for explaining the concept of the light emission profile. As shown in the upper part of FIG. 8, the pre-captured image is captured by causing the illumination unit 22 to scan and move in the scanning movement direction Ds to pass above the well W. The middle part of the figure shows an example of the luminance distribution obtained from the pre-captured image, the solid line shows the luminance distribution at a position directly below the locus of the light emitting element 221a passing near the center of the well W, and the broken line shows the periphery of the well W. Luminance distributions at positions immediately below the locus of the light emitting element 221b passing through the vicinity are shown.

  The lower part of FIG. 8 shows the relationship (light emission profile) between the light emission amount of each light emitting element in the main scanning operation necessary for canceling such a luminance distribution and the position (scanning position) of the light emitting element in the scanning movement direction Ds. ing. A solid line indicates a light emission profile for the light emitting element 221a passing above the vicinity of the center of the well W, and a broken line indicates a light emission profile for the light emitting element 221b passing near the periphery of the well W.

  While making each light-emitting element 221 emit light with a constant light emission amount, at a position where an increase in luminance is seen in the pre-captured image, the light emission amount of the corresponding light-emitting element is reduced, resulting in incidence to the bottom surface of the well. It is possible to make the light amount distribution of the illumination light to be substantially uniform. Thus, a light emission profile can be created for each light emitting element 221 provided in the illumination unit 22 (or for each group of light emitting elements) based on the luminance distribution of the pre-captured image.

  Returning to FIG. 7, the description of the imaging operation of this embodiment will be continued. When the light emission profile for each light emitting element is created in this way, the main scanning operation by the one-dimensional imaging element 133 is executed while controlling the lighting amount of each light emitting element 221 on the basis of the light emission profile to adjust the light emission amount. Is acquired (step S208). At this time, the amount of light emitted from each light-emitting element 221 is adjusted in real time in synchronization with the scanning movement of the one-dimensional imaging element 133, so that imaging can be performed with a constant amount of light incident on the bottom surface of the well. Therefore, similarly to the first embodiment, it is possible to acquire an image without luminance unevenness caused by refraction of illumination light on the sample surface.

  FIG. 9 is a diagram showing a main part of a third embodiment of the imaging apparatus according to the present invention. This embodiment has a feature in the configuration of the illumination unit 32, and the configuration of the first embodiment or the second embodiment described above can be applied to other portions. Therefore, the characteristic part of this embodiment is mainly demonstrated here.

  The illumination unit 32 according to this embodiment includes a light source 321 that emits light, and a reflection mirror unit 322 that is disposed above the well W and reflects the light L31 emitted from the light source 321 to enter the well W. ing. The reflection mirror unit 322 is configured by arranging a large number of minute reflection mirrors 323 whose angles can be adjusted independently of each other. Each of the plurality of reflection mirrors 323 is angle-controlled by a mirror control unit 332 provided in the control unit 10. As such a reflection mirror unit 322, for example, a DMD (Digital Mirror Device) used in a display device can be used. Further, as the light source 321 and the reflection mirror unit 322, either a two-dimensional (surface light source) type as in the first embodiment or a one-dimensional (line light source) type as in the second embodiment can be used. is there. In the one-dimensional type, as in the second embodiment, at least a scanning movement in synchronization with the scanning movement of the imaging element of the reflection mirror unit 322 is required.

  In such a configuration, the reflection direction of the light incident from the light source 321 is changed for each position by the reflection mirror unit 322, thereby controlling the light amount distribution of the light L32 incident on the well W, and the amount of incident light on the well bottom surface. Uniformity can be achieved.

  FIG. 10 is a diagram showing the main part of a fourth embodiment of the imaging apparatus according to the present invention. In each of the above embodiments, a pre-captured image is acquired by performing a pre-scan operation prior to the main scan operation, and an irradiation light amount distribution is set from the luminance distribution. On the other hand, in the apparatus of the fourth embodiment, the pre-scan in each imaging is performed by setting the light amount distribution of the illumination light in advance by assuming the irradiation light amount unevenness caused by refraction on the sample surface. The operation is omitted. Such an embodiment is suitable when the variation of the surface state of the sample is small, for example, the viscosity of the fluid injected into the well W is low and the injection is automated.

  As shown in FIG. 10A, in the illumination unit 42 of this embodiment, a surface light source 42a having a substantially uniform light quantity distribution two-dimensionally is disposed above the well W. Between the surface light source 42a and the well W, a transmitted light amount adjusting member 42b on which a shading pattern as shown in FIG. The transmitted light amount adjusting member 42b is a sheet-like or flat plate-like member having optical transparency. As shown in FIG. 10B, the color is darker at the portion corresponding to the center of the well W, and the color is further away from this. It is getting thinner.

  Therefore, when the transmitted light amount adjusting member 42b is disposed above the well W, the light L41 emitted from the surface light source 42a has a substantially uniform light amount distribution, and passes through the transmitted light amount adjusting member 42b and enters the well W. In the light L42, a light amount distribution is given such that the light amount is small in the central portion of the well W and the light amount increases in the peripheral portion. Thereby, it is possible to increase the amount of incident light at the peripheral portion where incident light is difficult to reach compared to the central portion, and to bring the amount of light at the bottom of the well closer to a uniform one. At this time, it is desirable that the center of the light and shade pattern of the transmitted light amount adjusting member 42 b be easily aligned with the center of the well W. For example, alignment marks may be provided in advance on the transmitted light amount adjusting member 42b and the microplate M. Further, for example, the transmitted light amount adjusting member 42b may be formed as a lid that engages with the microplate M and covers the upper portion of the well W, so that the alignment is automatically performed by the engagement.

  FIG. 11 is a flowchart showing an example of a method for creating a transmitted light amount adjusting member in the fourth embodiment. First, a calibration sample for obtaining the light amount distribution of illumination light in the well is prepared (step S301). The calibration sample may be the sample itself that is the object of the imaging operation, or may be prepared separately by injecting the same type and amount of fluid into another well. Good. Using the calibration sample thus prepared, imaging is performed in the same manner as in the first embodiment, and a pre-captured image is acquired (step S302).

  Subsequently, with respect to the pre-captured image obtained in this way, a plurality of line segments that cross the well W are set (step S303), and set in the well region corresponding to the well W in the pre-captured image. The luminance value of each pixel located on each line segment is calculated from the image data (step S304).

  FIG. 12 is a diagram illustrating an example of setting a line segment. As shown in FIG. 12A, four line segments A-A ′, B-B ′, C-C ′, and D-D ′ are set for the image of one well W here. Each line segment is set at equiangular intervals so as to cross each other in the vicinity of the center of the well W. Therefore, in this example, the angle formed by the adjacent line segment is 45 degrees.

  FIG. 12B shows an example of a luminance profile obtained from a data string of luminance values of each pixel on one line segment (for example, line segment A-A ′). Corresponding to the fact that the amount of illumination light reaching the bottom surface of the well W is high in the central portion and low in the peripheral portion, the luminance of each pixel in the central portion of the well region WR corresponding to the well W is shown in FIG. On the other hand, the brightness of each pixel is relatively low at the peripheral portion of the well region WR.

  The luminance profile shown in FIG. 12B includes fine fluctuations and spike-like noise, but as described above, it is considered that the luminance change caused by the illumination light is more gradual. Therefore, the obtained luminance profile is smoothed (step S305). As the smoothing process, for example, a moving average process between adjacent data, a process of extracting an upper envelope of a waveform, or the like can be used.

  Next, the luminance distribution of the entire well W is calculated from the luminance profile thus obtained on each line segment (step S306). Here, a method of calculating the luminance value of each pixel in the well by interpolation calculation from the luminance profile on each line segment will be described, but it is of course possible to obtain the luminance distribution by the same method as in the first embodiment. . Also, the luminance distribution calculation method of the present embodiment can be applied to the first embodiment and the like.

  FIG. 13 is a diagram for explaining the calculation principle of the luminance value of the pixel in the well. In this method, the luminance value at an arbitrary point Q in the well W is calculated by interpolation from a known luminance profile. First, an XY plane with the center O of the well W as the origin is set. In the example of FIG. 13, the coordinate axes are set so that the line segment AA ′ and the line segment BB ′ orthogonal to each other match the X axis and the Y axis (more precisely, match the coordinate axes). These line segments are set in advance as described above).

  Then, of the previously set line segments, two that sandwich the point Q for calculating the luminance value are specified. In this specification, the rotation angle of the line segment OQ connecting the origin O and the point Q and the rotation angle of each line segment A-A ′ set in advance can be used. Specifically, when the rotation angle of the line segment OQ about the origin O from one axis, for example, the X axis, is θ, the line segments AA ′, BB ′, CC ′, Among DD ′, the line segment whose rotation angle value from the X axis with the origin O as the center is the closest to the angle θ and the line segment closest to the line segment are specified as the line segments sandwiching the point Q. In the example of FIG. 13, the line segment B-B 'and the line segment D-D' correspond to this. The rotation angles from the X axis around the origin O of each of the line segments B-B ′ and D-D ′ are represented by symbols α and β, respectively. The case where the point Q is a point on any line segment may be excluded. This is because the luminance value of the point Q has already been obtained when the above luminance profile is derived.

  Next, the points R and S having the same distance from the origin O as the points Q on the line segments B-B ′ and D-D ′ sandwiching the point Q are specified. The luminance values at these points R and S have already been obtained, and the values are Lr and Ls, respectively. Since the change in the amount of light in the well W due to the influence of the illumination light source and the meniscus is gradual, the luminance value changes continuously and gently on an arc passing through the points Q, R, and S with the origin O as the center. Can think. For example, if it is considered that the luminance value on the arc is proportional to the rotation angle of the radial radius around the origin O, the luminance value Lr at the point R on the line segment BB ′ and the line DD ′. The luminance value Lq at the point Q can be obtained from the luminance value Ls at the point S and the rotation angles α, β, θ including the line segment OQ as follows.

That is, the following simultaneous equations for variables m and n:
θ = mα + nβ
m + n = 1
And the calculated values of m and n are:
Lq = mLr + nLs
Assign to. Thereby, the luminance value Lq at an arbitrary point Q in the well W can be obtained. By performing the above calculation at each position in the well W, the luminance distribution in the entire well W can be obtained.

  Returning to FIG. 11, the description of the method of creating the transmitted light amount adjustment member will be continued. When the luminance distribution in the entire well W is calculated as described above, the density distribution of the transmitted light amount adjusting member for subsequently canceling the luminance distribution and obtaining uniform illumination conditions is calculated. That is, for each position in the well W, a relatively high density mask pattern is arranged in a region where high luminance is detected in the pre-captured image to limit the amount of light incident on the well W, while the low luminance well By arranging a low-density mask pattern in the region so that more light is incident, the light quantity distribution of the illumination light in the well can be made uniform.

  Specifically, by appropriately scaling the luminance value obtained at each position of the well W and adding an appropriate offset value as necessary, the density value of the mask pattern of the pixel corresponding to each position can be obtained. It is obtained (step S307). From the viewpoint of uniformizing the light quantity distribution of the illumination light in the well W, the relative density difference for each position is more important than the absolute value of the density value. That is, for the purpose of controlling the brightness of the entire image, an appropriate offset value may be added to the density value of each pixel obtained from the pre-captured image.

  Then, by creating a shading pattern having a density corresponding to the density value for each pixel thus obtained on the transparent sheet (step S308), for example, a transmitted light amount adjusting member 42b as shown in FIG. Created. The production of the light and shade pattern can be performed by, for example, ink jet printing. In addition, due to the combination of the well W and the fluidity of the sample, the characteristics are different for each well W, and it may be troublesome to individually prepare a correction light / dark pattern. In such a case, a plurality of cases having similar luminance distributions may be grouped, and a shading pattern based on the luminance distribution statistically calculated within the group may be obtained and used in common within the group. .

  FIG. 14 is a diagram showing a main part of fifth and sixth embodiments of the imaging apparatus according to the present invention. In the fifth embodiment shown in FIG. 14A, the irradiation light L51 from the illumination unit 52 having the light source 52a and the transmitted light amount adjustment unit 52b is reflected by the total reflection mirror 520, and the reflected light L52 enters the well W. To do. Such a configuration is substantially the same as that of the first embodiment except for the optical path, and the same effect as that of the first embodiment can be obtained.

  In addition, in the sixth embodiment shown in FIG. 14B, instead of arranging the imaging unit 13 below the well W as in the above embodiments, the imaging unit 613 is arranged above the well W. It is. More specifically, a half mirror 620 is provided above the well W, and an imaging unit 613 is provided above the half mirror 620. Then, the irradiation light L61 from the illumination unit 62 having the light source 62a and the transmitted light amount adjustment unit 62b is incident on the half mirror 620, and the reflected light L62 is incident on the well W. On the other hand, the light L 63 emitted upward from the well W is transmitted through the half mirror 620 and is incident on the imaging unit 613. Even in this case, as in the above embodiments, it is possible to irradiate the object in the well W with substantially uniform light and obtain an image with little luminance unevenness. Note that even if the illumination unit 62, the half mirror 620, and the imaging unit 613 are arranged below the microplate M, imaging is possible.

  As described above, in these embodiments, the microplate M corresponds to the “sample holding plate” of the present invention, and the well W corresponds to the “concave portion” of the present invention. The holder 11 functions as the “holding unit” of the present invention, while the imaging unit 13 functions as the “imaging unit” of the present invention. The one-dimensional image sensor 133 in the first and second embodiments corresponds to the “line sensor” of the present invention. Moreover, the illumination parts 12, 22, 32, 42, 52, and 62 in each embodiment function as the “light irradiation means” of the present invention. In addition, when the light emitting elements 221 in the second embodiment are grouped, each group corresponds to the “light emitting module” of the present invention.

  In the first to third embodiments, the control unit 10, more specifically, the shutter control unit 112 b in the first embodiment, the light source control unit 122 in the second embodiment, and the mirror control unit 332 in the third embodiment are provided. It functions as the “control means” of the invention, and in the fourth embodiment, the transmitted light amount adjustment member 42b functions as the “control means” of the present invention.

  Further, steps S101 and S102 in the flowchart of FIG. 4 correspond to the “preliminary imaging process” of the present invention, while steps S103 and S104 correspond to the “luminance distribution detection process” of the present invention. Steps S105 and S106 correspond to the “light quantity distribution setting step” of the present invention, and step S107 corresponds to the “image acquisition step” of the present invention.

  The present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, in the first embodiment, an arbitrary two-dimensional light amount distribution can be obtained by a combination of a surface light source and a liquid crystal shutter. However, as shown in the second embodiment, a large number of light emitting elements are used. Similarly, the light quantity distribution can be arbitrarily set by controlling lighting of each light emitting element individually using light sources arranged two-dimensionally.

  Conversely, also in the second embodiment, it is possible to create an arbitrary light amount distribution by a combination of a light source having a fixed light amount distribution and a transmitted light amount adjusting unit such as a liquid crystal shutter.

  Moreover, in the said 1st Embodiment, although the liquid crystal display panel with which the surface light source and the liquid-crystal shutter were integrated is used as an illumination part, a light source and a transmitted light amount adjustment part are comprised separately, and it is comprised. Also good. Further, a diffusing plate or the like for making the light amount distribution uniform may be further provided between the light source and the transmitted light amount adjusting unit.

  Further, although not particularly mentioned in the description of the above-described embodiment, it is only necessary to obtain a background image in which the light amount distribution of illumination light is reflected in the pre-scan operation for acquiring a pre-captured image. In this sense, the required resolution may be considerably lower than that in the main scanning operation. Therefore, in the pre-scan operation, the scanning speed of the image sensor is increased to shorten the processing time, while in the main scan operation, a high-resolution image is acquired by scanning the image sensor at a lower speed. It may be executed by switching the speed. Further, more uniform illumination conditions may be obtained by repeating the pre-scan operation and the adjustment of the light amount distribution based on the result a plurality of times.

  Based on the same idea, the pre-scan operation may be performed by switching the imaging target area, such as imaging a plurality of wells at once while the main scan operation imaging each well individually. Good.

  Further, when a color liquid crystal display panel is used as the transmitted light amount adjustment unit of the present invention, the following usage is possible by changing the transmission pattern for each color. For example, by changing the color of the illumination light in accordance with the color of the cell to be analyzed, an image in which the contrast of a specific type of cell is enhanced can be acquired.

  In addition, as the transmitted light amount adjusting member in the fourth embodiment, a liquid crystal shutter as shown in the first embodiment may be used. Further, as shown in the second embodiment, a predetermined light amount distribution may be created by changing the amount of light emitted from the light source.

  In addition, the imaging device of each of the above embodiments is a device that can perform analysis by various image processing on the captured image, but the present invention is also applied to a device that simply performs imaging. Is possible.

  Further, in each of the above-described embodiments, by managing the light amount distribution of light incident on the well W, uniform illumination light strikes the objects distributed in the well W. On the other hand, from the viewpoint of not causing luminance unevenness in the captured image, the light amount distribution is adjusted on the optical path until the light emitted from the well W enters the imaging unit, as described below. Is also possible.

  FIG. 15 is a diagram illustrating an example of adjusting the light amount distribution between the well and the imaging unit. In this example, an illumination unit 72 having an appropriate in-plane light quantity distribution is provided above a microplate M having a well W, and illumination light L71 is incident from above the well W. A transmitted light amount adjustment unit 720 similar to that used in each of the above embodiments is disposed immediately below the bottom surface of the well W, and an imaging unit 713 is provided below the same. In such a configuration, even if the light emitted from the well W has a light amount distribution due to nonuniform illumination light, the light that passes through the transmitted light amount adjustment unit 720 and enters the imaging unit 713 In L72, the non-uniformity is eliminated. Thereby, luminance unevenness in the captured image is suppressed.

  As described above, by adjusting the light amount distribution of the light finally incident on the imaging unit 713, it is possible to suppress the luminance unevenness of the image. Even in an imaging unit using a light-receiving element that has nonlinearity in sensitivity to light, it is possible to obtain an image with less influence of such nonlinearity by setting the luminance of the background portion of the object to a substantially uniform level. Is possible.

  Note that the transmitted light amount adjusting member 720 may be in close contact with the lower surface of the microplate M. In particular, when a sheet-like member on which the shading pattern shown in the fourth embodiment is formed in advance is used as the transmitted light amount adjusting unit 720. Alternatively, such a sheet may be attached to the lower surface of the microplate M. For example, a sheet in which a light and shade pattern is formed and previously integrated with the microplate M can be used.

  The present invention can be particularly suitably applied to a field that requires observation of a sample in which a fluid is injected into a recess, such as a well on a microplate used in the medical / biological science field. The application field is not limited to the medical / biological science field.

DESCRIPTION OF SYMBOLS 1 Imaging device 10 Control part (control means)
11 Holder (holding means)
13 Imaging unit (imaging means)
42b Transmitted light amount adjusting member (control means)
112b Shutter control unit (control means)
122 Light source control unit (control means)
133 One-dimensional image sensor (line sensor)
221 Light emitting element (light emitting module)
332 Mirror control unit (control means)
M microplate (sample holding plate)
S101, S102 Pre-imaging process S103, S104 Luminance distribution detection process S105, S106 Light intensity distribution setting process S107 Image acquisition process W well (recessed part)

Claims (16)

  1. Holding means for holding the sample holding plate formed with a recess capable of holding liquid in a substantially horizontal state;
    A light irradiating means for causing light to enter the surface of the sample formed by injecting a fluid into the recess from above the sample holding plate held by the holding means;
    Imaging means for imaging the sample and obtaining an image of the sample;
    Control means for controlling the light quantity distribution of light incident on the surface of the sample from the light irradiation means,
    The control means sets the light quantity distribution in a predetermined standard state in advance, and based on the luminance distribution in the background image in which the imaging means removes high frequency components of a predetermined spatial frequency or higher from the pre-captured image obtained by imaging the sample. And setting the light quantity distribution of the light incident on the surface of the sample,
    The imaging apparatus is characterized in that the imaging unit acquires an image of the sample under light irradiation with a light amount distribution set by the control unit.
  2.   The imaging apparatus according to claim 1, wherein the control unit increases the amount of incident light at a position where the luminance in the background image is lower.
  3. The light irradiation means includes a light source and a transmitted light amount adjustment unit that is disposed between the light source and the sample and can set a light transmission amount for each position, and the control unit includes the transmitted light amount adjustment unit. the imaging apparatus according to claim 1 or 2 for controlling.
  4. The imaging apparatus according to claim 3 , wherein the transmitted light amount adjustment unit can set different amounts of transmission for a plurality of wavelength components included in light from the light source.
  5. The transmitted light amount adjusting unit image pickup apparatus according to claim 3 or 4 is a liquid crystal shutter.
  6. The light irradiation unit includes a light source formed by arranging a plurality of light emitting modules, imaging apparatus according to claim 1 or 2, wherein said control means individually controls the light emission amount of the plurality of light emitting modules.
  7. The light irradiating means includes a light source, and a plurality of reflecting mirrors that reflect light from the light source to be incident on the surface of the sample, and whose reflection directions can be changed independently of each other, and the control means includes the the imaging apparatus according to claim 1 or 2 for controlling the reflection direction of each of the plurality of reflecting mirrors.
  8. The imaging means, the imaging apparatus according to any one of claims 1 to 7 having a line sensor for acquiring an image of the sample by relative scanning movement with respect to the sample holding plate.
  9. The imaging apparatus according to claim 8 , wherein the light irradiation unit moves relative to the sample holding plate integrally with the line sensor as the line sensor scans and moves with respect to the sample holding plate.
  10. The imaging device according to claim 9 , wherein the control unit changes a light amount distribution of light incident on the surface of the sample in synchronization with a relative movement of the light irradiation unit with respect to the sample holding plate.
  11. In an imaging method for imaging a sample formed by injecting a fluid into a recess provided in a sample holding plate,
    A pre-imaging step of irradiating light with a light intensity distribution in a predetermined standard state from above the sample holding plate held substantially horizontally toward the surface of the sample and capturing the sample to obtain a pre-captured image; ,
    A luminance distribution detection step of detecting a luminance distribution of a background image obtained by removing high frequency components of a predetermined spatial frequency or higher from the pre-captured image;
    A light amount distribution setting step for setting a light amount distribution of light incident on the sample based on the detection result in the detection step;
    An image acquisition method comprising: an image acquisition step of acquiring an image of the sample under light irradiation with the light amount distribution set in the light amount distribution setting step.
  12. The light intensity in the distribution setting step, the imaging method of claim 1 1, the luminance in the background image to increase the amount of incident light as a low position.
  13. The pre imaged by the step and the image acquisition step, an imaging method according to claim 1 1 or 1 2 line sensors are relatively scanning movement relative to the sample holding plate imaging the sample.
  14. Imaging according to the light irradiating means for irradiating light to the specimen, to claim 1 3 relatively moves with respect to the line sensor integrally with the sample holding plate with the scanning movement of said line sensor with respect to the sample holding plate Method.
  15. In the image acquisition step, the light amount distribution of light incident on the surface of the sample from the light irradiation unit is synchronized with the relative movement of the light irradiation unit with respect to the sample holding plate based on the setting in the light amount distribution step. the imaging method according to claim 1 4 for imaging while changing.
  16. The pre-imaging step, the luminance distribution detection step, the light amount distribution setting step, and the image acquisition step are executed for each of the samples held in each of the plurality of recesses provided on the sample holding plate. imaging method according to any one of claim 1 1 to 1 5.
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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016540237A (en) * 2013-12-06 2016-12-22 バクテリオスキャン エルティーディー Optical measurement of liquids with free surfaces.
JP6301194B2 (en) 2014-05-26 2018-03-28 浜松ホトニクス株式会社 Optical plate, light irradiation device, light measurement device, light irradiation method, and light measurement method
CN104092941A (en) * 2014-07-10 2014-10-08 深圳市得意自动化科技有限公司 Camera shooting method achieved through camera shooting elements
JP6389721B2 (en) * 2014-09-30 2018-09-12 株式会社Screenホールディングス Imaging apparatus and imaging method
JP6535494B2 (en) * 2015-03-31 2019-06-26 株式会社Screenホールディングス Imaging device, imaging method and culture vessel
JP6534294B2 (en) * 2015-04-30 2019-06-26 富士フイルム株式会社 Imaging apparatus and method, and imaging control program
CN105136684A (en) * 2015-08-14 2015-12-09 上海蓝怡科技股份有限公司 Multi-sample detection device and method
JP6577793B2 (en) * 2015-08-28 2019-09-18 株式会社Screenホールディングス Light regulating device and imaging method
JP6239562B2 (en) 2015-09-14 2017-11-29 株式会社東芝 Lighting device and bio information measuring device including the same
WO2018061131A1 (en) * 2016-09-28 2018-04-05 オリンパス株式会社 Cell status assessment device
KR20180046098A (en) * 2016-10-27 2018-05-08 삼성전자주식회사 Test Apparatus, Test System and Control Method of Test Apparatus
US20180164567A1 (en) * 2016-12-12 2018-06-14 Molecular Devices, Llc Trans-Illumination Imaging with an Array of Light Sources
WO2019069823A1 (en) 2017-10-03 2019-04-11 富士フイルム株式会社 Imaging device, method for actuating imaging device, and imaging control program
WO2020066959A1 (en) * 2018-09-25 2020-04-02 パイオニア株式会社 Optical sample, optical member, and optical device

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06148066A (en) 1992-11-06 1994-05-27 Suzuki Motor Corp Optical system correcting device for particle agglomeration inspecting device
JPH06174635A (en) 1992-12-08 1994-06-24 Suzuki Motor Corp Face emission source for agglomeration tester
US6718053B1 (en) 1996-11-27 2004-04-06 Chromavision Medical Systems, Inc. Method and apparatus for automated image analysis of biological specimens
JPH1137924A (en) * 1997-07-18 1999-02-12 Hitachi Koki Co Ltd Brightness correction method for aggregation image discriminating system
AT425448T (en) * 1998-05-16 2009-03-15 Applera Corp Optical device, especially for monitoring dna polymerase chain reactions
EP1177523B1 (en) 1999-04-13 2013-08-21 Chromavision Medical Systems, Inc. Histological reconstruction and automated image analysis
JP2001083090A (en) * 1999-09-10 2001-03-30 Fuji Photo Film Co Ltd Excitation light source for micro titer plate
AU2002228837B2 (en) 2000-11-03 2007-06-28 Cytyc Corporation Cytological imaging systems and methods
US7050620B2 (en) 2001-03-30 2006-05-23 Heckman Carol A Method of assaying shape and structural features in cells
US7120282B2 (en) 2003-01-29 2006-10-10 General Electric Company Method and apparatus for correcting digital X-ray images
US7129473B2 (en) 2003-05-16 2006-10-31 Olympus Corporation Optical image pickup apparatus for imaging living body tissue
JP4377171B2 (en) * 2003-07-15 2009-12-02 Tdk株式会社 Spatial light modulator
US7283654B2 (en) 2004-08-26 2007-10-16 Lumeniq, Inc. Dynamic contrast visualization (DCV)
US7718131B2 (en) 2005-07-06 2010-05-18 Genetix Limited Methods and apparatus for imaging and processing of samples in biological sample containers
EP1912072A1 (en) * 2005-08-03 2008-04-16 Olympus Corporation Mixing device and analysis device having the mixing device
WO2007074923A1 (en) * 2005-12-27 2007-07-05 Olympus Corporation Apparatus for measuring luminescence dose and method of measuring luminescence
US7516934B2 (en) 2006-02-24 2009-04-14 Bio-Rad Laboratories, Inc. Sample plate support of adjustable angular orientation
JP4869843B2 (en) 2006-09-06 2012-02-08 オリンパス株式会社 Cell image processing apparatus and cell image processing method
WO2008032096A2 (en) 2006-09-14 2008-03-20 Oxford Gene Technology Ip Limited Apparatus for imaging single molecules
US7692162B2 (en) * 2006-12-21 2010-04-06 Bio-Rad Laboratories, Inc. Imaging of two-dimensional arrays
US8351683B2 (en) * 2007-12-25 2013-01-08 Hitachi High-Technologies Corporation Inspection apparatus and inspection method
JP2010044004A (en) * 2008-08-18 2010-02-25 Nec Corp Apparatus, method and program for detecting transmitted light, and method of manufacturing sheet material
JP5745752B2 (en) 2009-05-21 2015-07-08 オリンパス株式会社 Cell screening method, control software used in the method, cell screening device, image analysis device, and cell screening system
WO2011048886A1 (en) * 2009-10-20 2011-04-28 オリンパスメディカルシステムズ株式会社 Fluorescence observation device
JP5543280B2 (en) * 2010-06-01 2014-07-09 キヤノン株式会社 Image processing apparatus and image processing method

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